Analysis of sugars by GC method

Discussions about methods and best practices across food quality, safety and authenticity testing.

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Hi all,

I am now try to anlayze the sugar (fructose, glucose, surcose, maltose, lactose and galactose) in food sample.
Apart from using HPLC method, I have found that there are GC method for sugar but need derivatization procedure such as trimethylsilylation.

Example of standard method is AOAC 971.18 Carbohydrate in Fruit Juices – Gas Chromatographic Method.
And, I am trying to follow the method and using GC-MSD for detection.

When I try to derivaztize the sugar individual standard(in solution, and blow dry by N2 prior to derivatization),
I have found there are 2 or more peak for the indivual sugar standard solution.

As mentioned as the following website,
https://sites.google.com/site/masonaco/ ... /gc-sugars
there are anomers of the sugar in form of solution.
As a result, I get a more than one peak (derivatized product) for the single compound.

Are this method is suitable for the quanititaive anlaysis?
If yes, how can I integrate the peak area (sum of the anomer peaks? only the D-sugar peaks?) for quantitation on standard and sample.

By the ways, the MS spectrum for the sugars are very similar and the retention time of the anomer peaks may overlap.
Is it enough for the structural confirmation?
I'm a bit interested as well. There was a presentation a few months ago at the midwest AOAC meeting about sugars analysis. Most did HPLC and had a fair ammount of trouble with matrix interference. There was one mention of a lab that did GC and they did pretty well but I couldn't get any info on the method.

From what I've seen most methods use silyation the classic method is Sweeley'S reagent which is a mix of HMDS and 1% I believe of TMCS. HMDS is a bit of a weaker silyating reagent. BSTFA or MSTFA are stronger and trimethylysilylimmidzole is more forgiving of residual water though it does not deriviatize any amino groups (which shouldn't be an issue).

The disagreement I've seen is whether or not to use hydroxylamine/methoxylamine to deriviatize the aldehyde or ketone or just use straight silylation.
http://www.shimadzu.eu/sites/default/fi ... _GC-MS.pdf

How about this app note using acetic anhydride and hydroxylammonium chloride. Only one peak per sugar.

added
Here is a clinchem article using microwaves to accelerate the reaction. Seems promising. Aldonitrile, and pentaacetate derivatives do not have the anomerization issues.
http://www.clinchem.org/content/52/2/334.full
The standard method to overcome the issue of double peaks due to anomers (of reducing sugars) is to reduce (NaHB4), then derivatize (TMS or OAc) and analyze by GC-MS.

Sucrose (non-reducing sugar) will not reduce, and will only give one peak.

JMB
That would work too. Sodium borohydride works even in aqueous solution. So you can extract with water then do the reduction to alchol, then blow down and do the acylation or silylation. It looks like the reduction takes a fair while though I bet it could be sped up by using a microwave or something like the clinchem article.

I haven't had much luck with silyation. I've tried it on acrylamide, mononucleotides and end up with nothing but junk, silyated impurities, large peak of the intact reagent, and the break down products. Also they tend to foul up the chromatographic system

Acetic anhydride is more benign.

The aldonitrile method's advantage is that if you are only looking for reducing sugars you can use an NPD as only reducing sugars get nitrilated though you can use an FID or MSD and see everything as well.

http://pubs.acs.org/doi/abs/10.1021/ja0 ... ode=jacsat

BTW I've been seeing a lot of methods lately using a microwave to accelerate the extraction or reaction. Does anyone know how to set up the microwave? It usually says something like 25% of exit power for 2 minutes. What settings on the microwave oven does that translate to?
Actually the reason for aldonitrile with hydroxylamine and acetic anhydride vs alditol acetate using NaBH4 is with alditol acetate fructose and glucose (nor any aldose vs ketoses) cannot be differentiated while with aldonitrile only glucose (aldoses) is nitrilated so it can be differentiated.


OTOH acetylation I believe only works for monosaccharides. Di's and tri's derivatives are not volatile enough for GC. Silyation works for disacharides but you are back to multiple peak problem. Even with oxime formation with hydroxylamine the TMS derivatives still have a syn and anti so 2 peaks.
My conclusions based on literature review is that the aldonitrile methd is best for monosaccarides particularly reducing sugars. You get one peak per sugar and the method is simple and clean for the GC. Unfortunately it doesn't render any di or poly saccarides volatile enough for GC

For di or polysaccarides TMS oximes are probably best. The hydroxylamine limits the peaks on reducing sugars to 2 syn and anti and the TMS ester are volatile up to trisaccarides.
I've been evaluating the aldonitrile method. It works very well for aldose monosaccharides and disaccharides. However it does not do ketoses ie fructose at all. I am trying a new method with ringlocked sugars with 1-methylimmidazole/DMSO and then acetic anhydrode however it looks to be a lot more messy than aldonitrile and the disaccharides get deleteriously affected as well compared to aldonitrile. I'd say use aldonitrile for aldoses and disaccarides (possibly trisaccarides with a high temp column) and use oxime acetates for ketoses (substitute methoxylamine for hydroxylamine) or TMS oximes or perhaps the 1MEI/DMSO procedure.
IN my final analysis I've decided the best way to do it is to dry it redissolve in 2% hydroxylamine in pyridine, heat for 90 deg for 30 minutes, cool, take one (small) portion and add a silylating reagent whichever one is cheapest BSTFA, MSTFA, HMDS+TMCS, TMSIM and use this alliquot for fructose or other ketoses and the main portion add acetic anhydride and make the paan (aldonitrile acetate) derivatives by heating for 90 deg for 30 minutes and use that for aldoses, sugar alcohols, and disaccharides.

similar to this method
http://online.analchem.cn:8080/fxhx/EN/ ... ct5781.htm

Fructose cis and trans oxime acetate seems to be a minor product of the hydroxylamine acetic anhydride reaction if it is there at all. Hydroxylamine + silyation reagent makes stable TMS oxime though the oxime can be cis or trans leading to two close peaks so just take the sum of the two.
mt_fong_01_13 wrote:
Hi all,

I am now try to anlayze the sugar (fructose, glucose, surcose, maltose, lactose and galactose) in food sample.
Apart from using HPLC method, I have found that there are GC method for sugar but need derivatization procedure such as trimethylsilylation.

Example of standard method is AOAC 971.18 Carbohydrate in Fruit Juices – Gas Chromatographic Method.
And, I am trying to follow the method and using GC-MSD for detection.

When I try to derivaztize the sugar individual standard(in solution, and blow dry by N2 prior to derivatization),
I have found there are 2 or more peak for the indivual sugar standard solution.

As mentioned as the following website,
https://sites.google.com/site/masonaco/ ... /gc-sugars
there are anomers of the sugar in form of solution.
As a result, I get a more than one peak (derivatized product) for the single compound.

Are this method is suitable for the quanititaive anlaysis?
If yes, how can I integrate the peak area (sum of the anomer peaks? only the D-sugar peaks?) for quantitation on standard and sample.

By the ways, the MS spectrum for the sugars are very similar and the retention time of the anomer peaks may overlap.
Is it enough for the structural confirmation?


Do you happen to have a HPLC method analyzing those sugars?
if you're looking for an HPLC method for sugars, ask the manufacturers. A lot has changed since the days of this rather elderly thread. There are now some Hilic methods around, and at least one column manufacturer sells a specific column optimized for analysis of sugars. Unfortunately I can't remember which rep showed me the chromatogram! Most will have a solution for sugars in their methods-database. Good luck!
My final method works pretty well for me though I don't do it very often

-evaporate in vac oven
-200ul hydroxylamine in pyridine
-exactly 30min at 90deg C (don't go longer)
cool and add 200ul acetic anhdride
-exactly 30min at 90deg C (don't go longer)
cool and add 0.5ml water and 0.5ml toluene
-centrifuge to allow organic layer to turn clear after acetic anhydride breaks down to acetic acid.

inject organic layer on a db-1/5/1701 column

It does monosacharides (a bit less sensitive for ketoses also prone to activity with them) sugar alcohols, and disaccharides.
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